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8th SEMINARLABORATORY METHODS BASED ON
ANTIGEN-ANTIBODY INTERACTIONS I
THE SENSITIVITY OF IMMUNOASSAYS
Sensitive methods:
• precise• expensive• usually used for verification
Less sensitive methods:
• give semiquantitative results• cheap• usually used for screening
IMMUNOAFFINITY CHROMATOGRAPHY
Separation/purification of antigens or antibodies from a mixture
column polymer beads
covalently boundantigen
affinity purified antibody : monoclonal antibodies which can be ordered from catalogues are also purified using this technique
AFFINITY PURIFICATION OF ANTIBODIES USING AN ANTIGEN-SORBENT COLUMN
1) Addition of antibodies to be purified
2) Binding3) Washing4) Elution
STEPS OF PURIFICATION
polymer bead
fixed antigen-specific Abs on the surface of the bead
column
PURIFICATION OF ANTIGENS1) Loading the antigen mixture
2) Binding
3) Washing
4) Elution
Purified antigens
ELISAEnzyme Linked Immune Sorbent Assay
ELISA plate
well
Enzyme Linked Immune Sorbent
Antibody conjugated with enzyme
enzyme
Antigen/antibody adsorbed to solid
surface
Enzyme activity is measured by the color reaction due to conversion of substrate
Similar principle applies to many other antibody-based detection methods
ENZYME ACTIVITY IN ELISA IS DIRECTLY PROPORTIONAL TO THE AMOUNT OF
IMMUNECOMPLEX PRESENT
BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Direct method Indirect method
Antigen
Primary antibodies
Label
Label
Secondary antibodies
Enzyme/anti-enzyme system
PAP – peroxidase / anti-peroxidaseAPAAP – alkaline phosphatase / anti- alkaline phosphatase
Antigen
Primaryantibody
Secondaryantibody
Enzyme-specific antibody, same isotype as the primary antibody
Enzyme
BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity )
Basic ABC
Antigen
Biotinylated antibody
Avidin
Avidin-enzyme complexes
Biotin-enzyme complex
Avidin-biotin enzyme
complexes
BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY
For antigens present at low concentration in complex biological samples
Removal of unbound materialBlocking free plastic surface with inert protein Removal of unbound protein
Addition of biotinylated antibody specific to a different epitope on target protein
Removal of unbound material
Addition of avidin-conjugated enzyme
Addition of substrate
Coating with Ag-specific „capture” antibody
Addition of antigen- containing solution
Removal of excess enzyme
STEPS OF COMBINED/’SANDWICH’ ELISA
STEPS OF BASIC INDIRECT ELISA Detection of antigen or specific antibody
Adsorption of antigen (coating)
Removal of excess antigen
Saturation of uncovered surface area with proteins
Removal of excess protein
Addition of Ag-specific antibodies
Addition of Secondary Ab conjugated with enzyme
Removal of excess antibody
Addition of chromogenic substrate
Removal of excess antibody
concentration
10
00
50
0
25
0
12
5
62
31
16
7.8
3.9
1.9
0.9
7
0.4
9
0.2
4
0.1
2
0.0
30
0.0
61
0.0
07
0.0
15
0.0
04
0
The sample with unknown concentration
OD
EQUAL ABSORBANCE = EQUAL CONCENTRATION
According to OD: it could be anyone
?
You should also dilute the unknown sample
This region could indicate the concentration
This region could indicate the concentration
PRACTICAL USE OF ELISA TECHNICS
Sandwich ELISAMeasuring the amount of a given
antigen (molecule)
• cytokines, hormones, drugs• viral/bacterial antigens –
diagnosis of infection• tumor antigens – diagnosis of
tumors / screening / follow-up
Indirect ELISAMeasuring the amount of antigen-
specific antibodies
• pathogen-specific antibodies – diagnosis of infection*
• isotype of antibodies – time course, monoclonal antibodies
• autoantibodies – diagnosis of autoimmune disorders
WESTERN BLOT (IMMUNOBLOT)
Steps:
1) Sample preparation (cells, tissues)
2) Gel electrophoresis
3) Blotting
4) Labeling (by primary and secondary antibodies)
5) Detection
Identification of defined components from protein mixtures by antigen specific antibodies
Anode(+)
Cathode(-)
Standard Protein sample
SDS-PAGE Membrane
Primary antibody binds to its epitope in the protein, then the labeled
secondary antibody binds to the primary antibody
Gel-electrophoresis Blotting LabelingLysis of sample Loading
X-ray film
WESTERN BLOT (IMMUNOBLOT)
IMMUNOPRECIPITATION
• Isolation and concentration of a particular protein from a protein mixture
• Detection of protein associations (e.g. members of receptor signalization)
CHROMATIN IMMUNOPRECIPITATION (ChIP)
Identification of molecules (mainly transcription factors) binding to a specific site of the DNA
Provides information about the link between signaling pathways and gene activation
Labeled antibodies added to fixed tissue sections detect the distribution of the chosen antigen within the tissue or within the cells
of a particular tissue
• Immunofluorescence• Fluorescent dye coupled to antibody
FITC – fluorescein isothiocyanate (green)PE – phycoerythrin (orange)
• Immunoenzyme method• enzyme-coupled antibody
P – peroxidase AP – alkaline phosphatase(Substrates converted into an insoluble compound)
IMMUNOHISTOCHEMISTRY
Tissue sample
Fixation
Section before staining
Freezing
Sectioning
IMMUNOHISTOCHEMISTRY
Secondary antibody Avidin
Cells
Slide
Primary antibody Biotin
Enzyme
X
Tissue sample
IMMUNOHISTOCHEMISTRY
Classical histochemistry Acute bronchopneumonia (hematoxylin-eozin staining)
Only few cell types could be identified
Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma)
Antinuclear (ANA) autoantibodies from the serum of a SLE patient can be visualized in cell culture (HEp-2) by indirect
fluorescent labeling (immunofluorescence)
A fixed and permeabilized skin fibroblast
Mitochondria F-actin Nucleus
Fixed and permeabilized pulmonary artery endothelial cell
Peroxisomes Mitochondria Nuclei